n. 1,,
`
`PCT
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`wonua flm oaommnou
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`
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`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(51) International Patent Classification 6 =
`A61L 27/00, C12M 3/04
`
`(11) International Publication Number:
`
`(43) International Pnblicafmn Date:
`
`wo 95/01310
`19 mum I995 (l9_m_95)
`
`Bromley. Kent BRI 2N'W (GB). PALMER, Debra [GB/GB];
`8
`bury, Tea
`, SP1 SPW (GB).
`
`('I4)Agent: Wl-[l'IE, Martin; Corporal: Patenlz 8: Trade Marks
`Dept. Smith & Nephew Group Research Centre, York
`Science Park. Hcatington. York ‘[01 SDF (GB).
`
`(21) International Application Number:
`(22) International Filing Date:
`
`PCI'lGB94l0l-655
`
`5 July 1994 (os.o7.94)
`
`(30)
`
`Dina:
`93140572
`931447 1 5
`93140564
`93145803
`93203529
`93203685
`
`7 July I993 (0107.93)
`7 July 1993 ((Tl.0‘7.93)
`'7 July l993 (0107.93)
`14 July 1993 04.07.93)
`2 Ocnober 1993 (0110.93)
`2 October 1993 (02.l0.93)
`
`GB
`GB
`GB
`GB
`GB
`GB
`
`(71) Applicant (for all designated sum except asp SMITH
`& N'EPl-[EW PDC [GB/GB]; 2 Temple Place, Victoria
`Embankment, London WC2R sap (GB).
`
`('72) Inventors; and
`
`With international search report.
`
`“mm
`B]:
`‘
`c.
`. Y
`SNH (GB). CARTER, Andrew. James [GBIGB]: 80 Debden
`R9:-d. Saffron Walden, Essex CB1] 4 AL (GB). SEARLE.
`RlCh8;tfk.ld1.n0[2GB/GB]; 12 Fairway Drive, Upper Poppie-
`tnn,
`Y
`GHE (GB). MATTTIEWS, Jane, Bridget
`[GB/GB]; 34 South Bank Avenue, York YOQ IDP (GB).
`KING. John. B. [GB/GB]; Well Cottage, Chiselhurst Road,
`
`(54) Title:
`
`IMPLANTABLE PROSTEEESIS, Kr!‘ AND DEVICE FOR MANUFACTURING THE SAME
`
`(51) Abstract
`
`An implantable prosthesis comprises a biocompmible. synthetic, substantially bioresorbable rnanix material seeded with fibroblasm.
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`507
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`MSD 1009
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`-a
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`FOR THE PURPOSES OF INFORMATION ONLY
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`Codes used tn identify Smcs party to the PC!‘ on the from page: of punphlets publishing intcmational
`applications under the PCI‘.
`Austria
`AIn1:nl.in
`B-Itbndnl
`Belgium
`Barth: Fuo
`Bulguin
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`BI?
`BG
`DJ
`BR
`BY
`CA
`CF
`CG
`(3
`CI
`CM
`CN
`(3
`CZ
`DE
`DK
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`E[
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`-1
`FR
`GA
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`§sas:=aaa:2:823::E%%§§§
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`§§g§§:s:=:::a:==aas2aa
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`IMPLANTABLE PROSTHESES, KIT AND DEVICE FOR MANUFACTURING THE SAME
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`The present invention relates to methods suitable for replacing
`
`or repairing broken or damaged connective tissue such as ligaments
`
`or tendons and to prostheses for use in such methods. Also
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`disclosed is a device for use in forming such prostheses, as well as
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`kits from which the prostheses can be formed.
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`it is known from United States Patent No. US5078744 to repair
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`damaged ligaments such as the anterior cruciate ligament (AC L) by
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`replacing part of the damaged ligament by a prosthetic ligament
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`comprising purified connective animal tendon or ligament tissue
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`fibres which are cross-linked and formed into groups of aligned
`fibres.
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`The most common method of repair or reconstruction of the
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`ACL is to implant a prosthetic graft comprising autogenous tissues.
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`Thus it is common surgical practice to harvest autogenous tissue
`eg. patellar tendon from the host and to form aprosthesis for
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`implantation.
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`A number of synthetic non-bioresorbable materials have been
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`used in the manufacture of prosthetic ligaments, the materials being
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`chosen for their affinity for supporting or encouraging the ingrowth
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`of fibroblasts, after irnplantation of the prosthesis.
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`According to the present invention there is provided an
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`implantable prosthesis which, in a form prior to implantation in a
`
`host, comprises a biocompatible, synthetic, substantially
`bioresorbable matrix material seeded with fibroblasts.
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`By "synthetic", is merely meant a material which is not used
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`naturally by the mammalian body in connective tissue repair or
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`which is not a chemically modified form of such amaterial. Thus this
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`term excludes collagen and artificially cross-linked collagen
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`matrixes (although, if desired, collagen can be used in addition to
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`the synthetic material).
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`The term "fibroblast" includes cells which are sometimes
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`referred to as fibroblast, fibrocyte, tenocyte or synovioctye cells.
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`This term also covers precursor cells to any of these cells.
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`By "substantially bioresorbable matrix material" is meant a
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`three dimensional structure for supporting fibroblasts (which may be
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`in the form of a scaffold, mesh or solid structure, for example) and
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`which, in the implanted prosthesis, degrades substantially over time
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`in a mammalian body, due to the chemicallbiological action of body
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`components (as opposed to simply breaking due to physical strain
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`on to the prosthesis). Desirably after the prosthesis has been
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`implanted in an adult human for five years (more preferably after
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`only one year's implantation) the bioresorbable material will have
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`degraded to such an extent so that it makes no substantial
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`contribution to the structural integrity of the prosthesis.
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`Preferably the matrix may additionally comprise one or more of
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`the following molecules: proteogiycans, glycosaminoglycans,
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`fibronectin or its active binding domain, or one or more growth
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`factors eg. bone morphogenetic protein (BMP) fibroblast growth
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`factor, angiogensis factor or other stimulatory factors.
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`In a further embodiment of the invention, the prosthesis or part
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`thereof (e.g. area(s) of the prosthesis which will come into contact
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`with bone after implantation), may be impregnated with
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`osteoinductive or osteoconductive agents, to enable more easy
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`infiltration by bone cells. Examples of suitable osteoinductive
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`materials susceptible to infiltration include hydroxyapatite, freeze-
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`dried or demineralised bone, growth factors (e.g. bone
`
`morphogenetic protein) etc. impregnation may suitably be just
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`before implantation of the prosthesis. Aptly such materials are
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`incorporated into ends of the prosthesis.
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`In a further embodiment of the present invention, there is
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`provided a method of repairing or replacing damaged connective
`
`tissue in a human or non-human animal comprising the steps of:
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`incubating a biocompatible, synthetic, substantially bioresorbable
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`matrix material in the presence of a suitable culture medium and of
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`fibroblasts under suitable conditions for fibroblast seeding on or in
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`the matrix and thereafter implanting the seeded matrix into a host.
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`Examples of suitable substantially bioresorbable synthetic
`
`polymers include polylactide (PLA), polyglycolide (PGA).
`
`polydioxanone, poly caprolactone (PCL), polyhydroxybutyrate (ICI
`
`BIOPOLW), polyhydroxybutyrate-co-hydroxyvalerate (ICI
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`BIOPOLW‘), polyanhydrides, polyorthoesters, potyorthocarbonates,
`
`polyaminocarbonates, polytrimethylene carbonate and co-polymers
`
`incorporating monomers from which the aforesaid polymers can be
`fanned.
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`When the prosthesis according to the present invention
`
`comprises a copolymer, the copolymer may incorporate
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`hydroxyvalerate and hydroxybutyrate monomers. In such
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`copolymers the amount of hydroxyvalerate present may be from 1 to
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`47% mol. Other particularly suitable copolymers are PLAIPGA and
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`PLAIPCL copolymers.
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`Composites of a plurality the above substantially bioresorbable
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`materials may also be suitable as or as part of the matrix material.
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`The matrix be fabricated of two or more distinct materials (eg.
`
`distinct fibre types) with different degradation rates, providing a two
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`or more phase loss of mechanical properties with time. Also, the
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`different fibre types may possess different mechanical properties.
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`For example. highly extendable fibres may be combined with less
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`extendable fibres. The matrix may be designed to elongate to a
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`specified extent before the less extendable fibres prevent further
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`extension. This design may be advantageous in exposing the cells
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`to limited and controlled strain while protecting against damage to
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`the forming tissue. For example, polycaprolactone fibres have a
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`lower Young's modulus than polylactide fibres.
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`Furthermore, one polymer may be coated with another
`
`polymer. This is advantageous where the material of choice on the
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`basis of mechanical properties is not necessarily the material of
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`choice for cell culture (unless it is modified). Here a more
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`biocompatible polymer may be used to coat a less biocompatible
`base material. For example, polyiactide provides a better substrate
`
`for fibroblast proliferation than polycaprolactone. Potycaprolactone
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`fibres could be coated with polylactide to improve compatibility with
`fibroblasts.
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`As indicated above, copolymeric materials may be used. This
`can be advantageous where the copolymers possess degradation
`rates intermediate between the rates of the homopolymers of which
`
`they are composed. Therefore, the degradation rate may be
`
`controlled by controlling the composition of the copolymer. Also,
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`production of copolymer fibres by fibre spinning or extrusion may
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`yield fibres with mechanical properties superior to those of
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`homopolymers. Polylactide-Polyglycolide copolymers are good
`
`examples of both of these points.
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`Suitable fibroblasts for use in seeding the matrix may be
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`autogenic fibroblasts, allogenic fibroblasts or xenogenic fibroblasts.
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`Preferably, the fibroblasts are autogenic. The fibroblasts may
`
`originate from for example the dennis, tendons or ligaments. The
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`fibroblasts forvuse in seeding the matrix may comprise a mixture of
`
`one or more of the above types of fibroblasts. Where the fibroblasts
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`are autogenic, it is preferable to isolate them from the dermis, as
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`this avoids the need for extensive invasive surgery.
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`The fibroblasts may be obtained according to any suitable
`
`method. A preferred method is by carrying out a skin biopsy.
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`_ The matrix material may be seeded with fibroblasts by placing
`
`the matrix in a culture vessel containing an appropriate culture
`
`medium (e.g. DMEM), in the presence of fibroblasts and incubating
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`under cell culture conditions. The fibroblasts may be suspended in
`the culture medium and the resultant suspension added to the
`culture vessel either before or after addition of the matrix. The
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`number of fibroblastslml of medium may be varied according to the
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`degree of seeding it is desired to establish.
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`The prosthesis of the present invention may be used to either
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`partially or totally replace a damaged ligament, tendon, cornea,
`
`dermis, dura (or other body part comprising connective tissue).
`
`Where the damage is substantial. the damaged ligament or tendon
`
`may be totally surgically replaced by the prosthesis. Where the
`
`damage is less substantial the matrix may be designed so as to be
`
`joined (e.g. by suturing) to the existing damaged ligament or tendon.
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`The matrix may be designed according to any one of a number
`
`of possibilities. Aptly the matrix is a fibrous structure. It may have
`
`loops or other structures at each end for aiding fixation to bone
`
`(using for example either the "two tunnel'’ or the "over-the-top"
`
`technique). It may be formed by any appropriate technique - e.g.
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`braiding, knitting, weaving, crocheting etc. The matrix is desirably in
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`elongate form and is preferably flexible.
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`The device may closely mimic the natural structure and fixation
`
`- of the ligament or tendon. For example, for ACL reconstruction, the
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`device could be composed of a hierarchy of fibres bundled together
`
`in fasicular units, passing directly from the femur to the tibia or
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`taking a spiral path around the axis of the device. Fixation may be to
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`the natural fixation areas of the ligament or tendon. Any appropriate
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`fixation means may be used (e.g. screws, nails, staples or sutines).
`The fixation means may itself be bioresorbable, for example it may
`be formed of polyhydroxybutyrate.
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`The present invention further provides a kit for forming the
`
`prosthesis of the present invention comprising a synthetic
`
`biocompatible matrix material and a source of fibroblasts.
`
`On incubation under suitable conditions, the fibroblasts will
`
`grow on and/or in the matrix. thus producing a matrix seeded with
`fibroblasts.
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`The kit may additionally comprise a suitable medium for the
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`proliferation of fibroblasts.
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`Ideally the kit is presented in a sterile package. Alternatively
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`the pans of the kit may be sterilised just before use. Prior to
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`implantation, the components of the kit can be incubated together
`
`under appropriate culture conditions as above described to allow
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`the fibroblasts to seed the prosthesis.
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`The fibroblasts may be in any suitable form ready for use.
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`Thus aptly the fibroblasts may be cryopreserved.
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`The matrix, or componentslprecursors thereof, may be
`
`provided in Iyophilised form.
`i
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`In a preferred embodiment, the present invention comprises,
`
`an implantable prosthesis which in a form prior to implantation
`
`comprises a biocompatible synthetic substantially bioresorbable
`
`matrix material having a polymeric gel in intimate contact therewith,
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`20
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`the gel having fibroblasts dispersed therein. This is advantageous in
`
`that the gel can support the cells in a true three-dimensional
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`arrangement rather than merely supporting a monolayer on the
`
`surface of a material. The environment closely mimics the natural
`
`physiological environment of the cells. Also, incorporation of cells in
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`a gel can provide for even cell distribution, preventing cells from
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`pooling which might otherwise occur due to gravitational influence.
`
`The present invention provides a method of repairing or
`
`repiacing connective tissue in a human or other animal, comprising
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`the steps of: incubating a biocompatible matrix material in the
`
`presence of a gel-fonning composition and of fibroblasts under
`
`suitable conditions to fonn a prosthesis comprising a matrix
`
`contacting a polymeric gel, the gel having fibroblasts dispersed
`
`therein, and thereafter implanting the prosthesis into a host.
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`Suitable gel forming compositions include collagen gel forming
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`compositions and fibrin gel fanning compositions.
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`Fibrobtasts in a collagen gel are capable of utilising the
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`collagen and reorganising it. Under an appropriate mechanical
`
`stimulus they are capable of reorganising the fibrils into non-
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`randomly orientated, organised structures resembling the natural
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`ultrastructure of ligament and tendons. A mechanical stimulus may
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`be the prevention of gel contraction which would otherwise occur
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`over time by fixing the gel at two points. The matrix may be
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`designed to achieve this. Alternatively, the gel on or in the matrix
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`10 may be exposed to applied strain using a mechanised straining
`
`device to stimulate fibroblast alignment.
`
`The method may comprise an additional step of incubating a
`
`gel-contacting matrix under suitable conditions for fibroblast
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`proliferation in the gel and thereafter implanting the matrix into a
`host.
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`In the preferred embodiment of the present invention. the
`
`matrix is seeded by means of incubating the matrix in the presence
`of a suitable culture medium, a gel-forming composition and the
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`20
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`fibroblasts to be seeded. An appropriate agent for causing gelation
`
`of the gel forming composition may also be used, if necessary.
`
`Seeding the matrix in the presence of a gel-fonning
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`composition, fibroblasts (and a gelling agent, if required) results in a
`
`gel—coated or filled matrix. the gel having fibroblasts dispersed
`
`therein. The gel can be formed by the interaction of the gelling
`
`agent and the gel-forming composition. A preferred gel is a collagen
`
`get. A Type I,
`
`II or Ill collagen solution may be prepared using an
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`appropriate source of collagen. Thus for example a Type I collagen
`
`solution may be prepared from dermis (Type I collagen forms up to
`
`70% of extracellular protein found in skin) as above described.
`
`Alternatively a Type I collagen solution may be prepared tendons,
`
`e.g. rat or bovine tendons, which comprise almost exclusively Type
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`1 collagen. The collagen may be extracted according to any of the
`standard" methods known by those skilled in the art.
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`There are a number of suitable ways of incorporating the gel in
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`or on the matrix. For example. the matrix may be suspended in such
`
`a manner that the gel-forming solution (optionally comprising
`
`fibroblasts) completely surrounds the matrix. A mould which
`surrounds the matrix may be used. Centrifugation or suction may
`
`alternatively be used to direct gel towards the matrix.
`
`A kit for use in forming the prosthesis of the preferred
`
`embodiment can comprise a biocompatible matrix, a gel-forming
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`composition and a source of fibroblasts.
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`Alternatively the kit may comprise a biocompatible matrix
`
`having a coating comprising a polymeric gel andlor having a
`
`polymeric gel incorporated therein and a source of fibrobtasts. On
`incubation under suitable conditions the fibroblasts can invade the
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`gel, thus producing a matrix bearing a gel having fibroblasts therein.
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`The prosthesis of the present invention offers an advantage
`
`over previously known prostheses which were designed to enhance
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`ingrowth of fibroblasts after implantation and act as a scaffold
`
`through which fibroblasts can grow and form a new ligament, since it
`
`comprises fibroblasts prior to implantation. Thus the damaged tissue
`
`may be replaced by a prosthesis comprising viable fibroblasts which
`
`may be replicating. The fibroblasts may already substantially be
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`aligned on implantation or at least oriented in a non-random
`
`manner. This process is speedier than previously known methods
`
`which rely on infiltration of prostheses by fibroblasts after
`
`implantation. It will be clear that the prosthesis may be implanted
`
`after an initial predetermined incubation period timed to result in
`
`seeding of the prosthesis with fibroblasts. Alternatively the
`
`prosthesis may be incubated for a longer incubation period than the
`
`initial incubation period so that the fibroblasts will be replicating and
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`will have already started to secrete collagen fibrils when the
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`prosthesis is implanted.
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`The prosthesis and method of the present invention offers
`other advantages over the common surgical practice or harvesting
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`host patellar tendon in that it avoids the need for carrying out an
`
`extensive surgical operation to harvest the tendon. A simple skin
`
`biopsy (a standard procedure which does not result in substantial
`
`scarring) can be used to obtain fibroblasts which can then be
`
`proliferated in culture. In addition. the prosthesis can be designed to
`
`optimise fibroblast orientation. The cumulative effect of these
`
`advantages can result in a reduction in the length of a hospital stay.
`
`in one preferred embodiment of the present invention, where a
`
`collagen gel contacts the matrix, the prosthesis of the present
`
`invention provides a source of collagen which can be used by the
`
`fibroblasts. The collagen in the gel is preferably in a non-cross-
`Iinked fonn.
`
`In another preferred embodiment of the present invention a
`
`fibrin get is used.
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`According to a further aspect of the present invention there is
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`provided a device for culturing cells for use in fonning a prosthesis
`
`according to the present invention, comprising a chamber for
`
`maintaining fibroblasts in a viable condition. the chamber being
`
`provided with means for releasably securing the matrix material and
`means adapted for applying strain to the matrix material along a
`
`single axis only. Such a device can be included in a kit as aforesaid.
`
`lf several straining means are present, the device of the
`
`present invention can apply strain to a plurality of samples at any
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`one time. Thus the device may have one or more chambers adapted
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`to retain a culture medium and may be provided with means for
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`releasably securing a plurality of matrix materials. This may be
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`don_e simultaneously. Each of several chambers may be provided
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`with means for releasably securing a plurality of matrix materials.
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`Alternatively each chamber may be provided with means for
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`releasably securing a single matrix material.
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`A chamber may be pennanently fixed within the device.
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`Alternatively the chamber may be releasably fixed so that it may be
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`removed from the device as desired, e.g. the chamber(s) may be
`
`removed to facilitate the securing and release of the matrix material.
`
`A chamber may be made from any material which may be
`
`sterilised by suitable methods of sterilisation, e.g. gamma
`
`irradiation, steam sterilisation or ethylene oxide (ETO) sterilisation.
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`A chamber may have any desirable shape and size. Suitably
`
`the chamber may be cylindrical, cuboid or spherical. The
`
`dimensions of the chamber should be such that they enable the
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`matrix material to be secured and to be subsequently extended on
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`the application of strain. The device of the present invention can be
`
`adapted to apply a strain which causes e.g. up to 100% extension of
`
`the matrix material (relative to the material in unextended fonn).
`
`Generally speaking however, an extension of up to 10%, or of up to
`
`5% may be sufflcient.
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`Thus the dimensions of the chamber can be such that they
`
`enable the matrix material to be extended to the desired level.
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`The dimensions of a chamber are desirably such that the
`chamber has a capacity of up to 75cm3, e.g. up to 50cm3. The
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`chamber may be made from any suitable material, e.g. stainless
`
`steel or Perspex“ material. Preferably the material is autoclavable
`to facilitate sterilisation.
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`Preferably the chamber is provided with a transparent or
`translucent window to enable the matrix material to be viewed
`
`during the time of culture. Examples of suitable materials include
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`glass and polymethylmethacrylate (PERSPEX).
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`The chamber may comprise a closure, which may be
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`removably or hingedly mounted to allow access to the inside of the
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`chamber. Thus for example the chamber may comprise a glass
`
`cylinder wherein at least one of the ends is removable. Aptly the
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`chamber may be collapsible or telescopic.
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`The chamber is desirably thennostatically controlled and may
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`be heated via a water jacket or other heating means. It may be
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`provided with various sensors e.g. sensors of the C02 content
`within a head space of the chamber. A CO2 source may also be
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`provided.
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`The matrix material may be releasably secured by securing
`means within the chamber.
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`The means for applying strain may compare two elements
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`which are movable within the chamber so that the spacing between
`
`the elements can be varied. Alternatively one element may be
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`movable but the other may be fixed.
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`The securing means may be any suitable means for releasably
`
`securing the matrix sample. The design of the securing means
`
`depends upon the design of the ends of the matrix material. Thus for
`
`example where the matrix material comprises looped ends the
`
`securing means may comprise a pair of clips or hooks. Suitably the
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`securing means may comprise for example a chuck or a lathe or
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`jaws which may screw together or be held by springs. Aptly the
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`securing means may be in the form of a slot or other opening such
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`that the ends of the matrix material are designed to fit thereon. Thus
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`for example the ends of the matrix material may be embedded in a
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`resin which may be retained in a slot. The opposite arrangement
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`can be used in which the opening is in the matrix material and the
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`securing means fit therein. A yet further way of releasably securing
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`the matrix material is to provide a spool, which may be generally
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`cylindrical, about which matrix material can be wrapped and held in
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`position by friction. The spool may be held in place by a gripping
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`device. Two such spools may be provided - one for each of two
`
`gripping devices.
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`The matrix material to which strain is to be applied by the
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`device of the present invention may comprise any suitable material
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`for supporting viable cells. Cells may be present in or along the
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`entire length of the matrix material. Alternatively part of the matrix
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`material. e.g. the ends thereof, may have no cells. The cells may be
`applied to the matrix material either before or after the matrix
`material has been secured under extension. It is preferable however
`to apply the cells to the material before securing the material under
`extension in the chamber.
`
`The matrix material may be designed in the form of a
`prosthetic ligament or tendon. Where for example the matrix
`material is in the fonn of a prosthetic ligament, the ligament when
`unstrained is preferably in the range of from 1 to 30cm long. The
`matrix material should be chosen so that it is suitable for
`withstanding the magnitudes of strain which it will be subjected to on
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`implantation, e.g. in the knee.
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`The means for applying strain may act by pulling both ends or
`one end of the matrix material, resulting in an extension of the
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`matrix material. This may be done by various means, e.g.
`mechanical, electrochemical, electrical, piezoelectric, pneumatic,
`hydraulic or other means. The matrix material may be releasably
`attached to a stationary element at one end of the chamber and the
`opposing end of the matrix material may be attached to a tension
`applying member (for example a winding device). Suitably strain
`may be applied to the matrix material by means of a diaphragm, one
`side of the diaphragm lying within the chamber and the opposing
`side lying outside the chamber. A pivotally mounted lever may be
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`used to apply strain.
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`The present invention also provides a method of culturing cells
`under strain which method comprises the steps of releasably
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`securing a matrix material having viable cells in intimate contact
`therewith in a chamber, the chamber comprising an adequate
`
`amount of culture medium to cover the matrix and cells, and
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`applying strain to the matrix material along a single axis.
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`Materials suitable for use in a matrix of the present invention can be
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`assessed as exemplified below:-
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`Assessment of Materials
`
`in order to make an initial assessment of suitable materials for
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`supporting fibroblast growth, various materials were obtained (which
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`are not to be construed as limiting), as indicated in Table 1 below,
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`and moulded into films for 5min at 2.5 Tons at the following
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`temperatures: Polylactide 170°C; polyglycolide, 245°C;
`
`polyhydroxybutyrate, 185°C; and polycaprolactone, 65°C.
`
`Table 1
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`15 Material
`
`Supplier
`
`Polylactide
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`Medisorb, Cincinnati, Ohio, USA
`
`Polyglycolide
`
`Medisorb, Cincinnati, Ohio, USA
`
`Polyhydroxybutyrate
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`Goodfellows, Cambridge, UK 1c
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`Polycaprolactone
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`Birmingham Polymers Inc.
`
`Alabama, USA
`
`fig
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`1a
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`1b
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`1d
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`Fibroblasts were seeded onto the surfaces of these materials
`
`at a density of 1 x 104 cellslcmz of material and incubated for 3
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`days under culture conditions.
`
`After the incubation period, photomicrographs were taken of
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`the cell-seeded samples. These are shown in Figs. 1a to 1d for the
`
`samples indicated in Table 1 above (photocopies of all of the
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`photographs provided for this application are provided immediately
`
`after the relevant photographs).
`
`Fibroblasts can be seen to be well adhered to the surfaces of
`
`all of the materials and to exhibit the morphology typical of healthy
`cultural fibroblasts.
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`One sample of Fibroblasts was grown on polylactide as
`described above apart from the fact that a longer (16 day) culture
`
`period was used.
`
`After this period the cells were stained with a viable stain
`(calcein AM (2uM)) and visualised by fluorescence microscopy
`using a fluorescein filter. A confluent monolayer of viable cells was
`observed, showing that polylactide is capable of supporting viable
`
`fibroblasts for extended periods of culture.
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`10
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`Figure 2 is a graph showing the relative rate of proliferation of
`
`fibroblasts on tour examples of bioresorbable synthetic materials:
`
`polylactide (PLA); polyglycolide (PGA) polyhydroxybutyrate (PHB)
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`and polycaprolactone (PCL) (all as described above) in comparison
`with a tissue culture treated polystyrene (TCP) control (since TCP is
`
`known to support good fibroblast growth).
`
`Figures 2a) to e) show each of these materials on a single
`graph (for each of reference). Cells were seeded at 1 X104 ce|ls.cm'
`2 in triplicate and the rate of proliferation detennined by measuring
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`the uptake of tritiated thymidine into cellular DNA at timepoints up to
`
`7 days after incubation using standard celi culture techniques. The
`
`medium was changed at 2, 4 and 6 days. The points represent the
`
`mean of three determinations and the error bars represent the
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`range. All polymers supported fibroblast proliferation.
`
`Figure 3 is a photomicrograph of fibroblasts embedded within a
`
`three dimensional collagen gel after 15 days of culture. The cell-
`
`seeded gel was prepared as described in example 2 (which _will be
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`30
`
`described later) apart from the fact that it was not used to contact a
`
`matrix. The gel provides a three-dimensional structure in which the
`
`cells are embedded and can form interactions with collagen
`
`molecules via membrane integrin receptors. The cells are randomly
`
`arranged, exhibit long processes and are capable of reorganising
`collagen fibrils within the gel.
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`Figure 4 is a photomicrograph of fibroblasts embedded within a
`three dimensional collagen gel as described for Fig. 3 above, apart
`
`from the fact that the gel has now been constrained from contracting
`
`in one direction by two stainless steel pegs glued to a culture dish
`with a tissue culture compatible adhesive. The cells are arranged in
`a highly orientated fashion, their long axes being parallel to the axis
`between the contraining pegs. The collagen fibrils align along the
`
`same axis. This effect is due to the pegs preventing the gel
`contracting, as would otherwise occur in the presence of fibroblasts
`in culture.
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`The following examples, which are not to be construed as
`
`limiting, illustrate how various cell-seeded matrixes can be
`
`produced.
`
`Example 1 : Preparation of a fibroblast seeded golylactide matrix
`prosthesis
`
`a) Preparation of Cells
`
`A biopsy is washed three times in phosphate buffered saline
`
`(PBS), and rinsed in 70% alcohol. The rinsed biopsy is then dipped
`
`into Dulbeccds Modified Essential Medium (DMEM) and incubated
`at 37°C for 24 hours. After incubation, the biopsy is cut into small
`
`pieces under PBS. The cut pieces are transferred to a 50mm petri
`
`dish, containing about 5ml of collagenase solution to allow
`digestion. The epidermal sheets are removed from the collagenase
`
`solution. The resultant solution is centrifuged. The fibroblast cell
`
`pellet is resuspended in DMEM and thereafter seeded in a 35mm
`
`petri dish using DMEM. The cells may be confluent in from 2-4 days.
`
`Thereafter the cells may be cultured to provide an appropriate
`
`quantity of fibroblasts for seeding the matrix.
`
`A suitable medium for culturing the isolated fibroblasts may
`
`comprise DMEM which may be supplemented with the following:
`
`glutamine, foetal calf serum, non essential amino acids and
`antibiotics. In addition the medium may have a buffering agent such
`as bicarbonate.
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`4
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`b) Pregaration of matrix material
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`25
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`' A polylactide matrix material suitable for use in a prosthesis for
`
`replacing a ligament can be prepared by obtaining polylactide fibres
`
`and then braiding them to fonn a braid of appropriate dimensions to
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`35
`
`replace the ligament.
`
`Polylactide fibres can be obtained by extrusion, fibre spinning,
`
`melt-spinning, drawing, heat annealing etc.
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`Braiding of the fibres can be done by standard braiding
`
`techniques, the length and thickness of the braid, number of fibres
`
`present and diameter of fibres present being selected to form a
`
`braid with appropriate properties.
`
`The ends of the device are constructed in a suitable way to aid
`
`fixation of the device by a screw or other fixation means. This is
`
`done by fonning eyelets at the ends.
`
`c) Seeding of matrix material with cells
`
`The braided device is incubated in a medium containing 10%
`
`vlv serum for 24 hours and is then seeded with cells by pipetting a
`
`cell suspension over the surface of the matrix material until the lat